The present invention relates to the field of aquaculture. More particularly, the present invention relates to the field of the therapeutic treatment of parasitic infections in fish. More particularly to the use of photoactive dyes such as Phloxine B as a method of controlling protozoan infections in fish populations. More particularly, the invention relates to the use of photoactive dyes such as Phloxine B for the treatment and/or prevention and of parasitic diseases in fish populations such as the disease Ick which is caused by the infection of fish with the external protozoan, Ichthyophthirius multifiliis. 
Aquaculture in the United States represents a relatively small segment of agricultural production, however the industry is relatively young and growing rapidly. Per capita consumption of fish food products in the United States has increased more than 50 percent since 1970. Furthermore, ornamental fish distribution has more than doubled since 1986. Food fish and ornamental fish markets combined contribute nearly $2 billion per year to the retail United-States economy. It has been estimated that these trends will continue in the new millenium.
Although the demand for food fish and ornamental fish products has increased, natural fish harvest populations have not increased and, in many cases, have declined. As such, aquaculture of fish species has increased to meet consumer demand. Among food fish, catfish, salmonid, and cichlid species dominate commercial aquaculture in the US. Catfish aquaculture, estimated at nearly $770 million in farm sales for 1999, accounts for about half of total annual US production. Among ornamental species, guppies, swordtails, and mollies dominate the ornamental fish market in the US. Ornamental aquaculture in Florida alone is valued at over $52 million.
Aquaculture of fish has been plagued by disease since its inception. Disease outbreaks are particularly prevalent when culturing large numbers of fish in crowded conditions. Many parasites and diseases can spread and quickly kill entire fish populations. Although the economic impact of infectious diseases is difficult to determine, it is estimated that annual losses in the US catfish industry may exceed $20 million.
A majority of fish losses in the aquaculture industry can be attributed to protozoan infections. Protozoans can infect both external and internal portions of the fish including the gills, fins, skin, and digestive organs. External protozoa of major concern to aquaculturists include members of the genus Costia, Chilodon, Scyphidia, Trichodina, Epistylis, Carchesium, and Trichophrya. The external ciliate, Ichthyophthirius multifiliis, causes white spot disease known as Ick or Ich. Ick is difficult to control and is often observed in crowded cultures of catfish and warm-water aquarium fish. Characteristic signs of Ick infection include the presence of grayish-white warts on the external surfaces of the fish and behavior changes such as flashing, jumping, or thrashing erratically in the water.
The life cycle of I. multifiliis is indirect. An adult stage called a trophozoite invades the skin or gills of fish and feeds directly on tissue fluids. Trophozoites, the form of Ick found on fish, are large protozoans and can easily be distinguished by light microscopy. Trophozoites are identified by a clearly visible C-shaped nucleus. After several days, depending on water temperature, the trophozoite releases from the fish, encysts, and undergoes fission to produce the next stage of its life cycle. After the cyst ruptures, an infective form called a tomite is released. As many as 2,000 tomites can be produced from a single cyst. Tomites are highly ciliated, pear-shaped (30-45 nm diameter), and are actively mobile in seeking out a new host. Prior therapeutic efforts to control Ick infections have focused on treating free-swimming tomites and unattached trophozoites. These therapeutic treatments are ineffective in controlling Ick trophozoites once they have attached to the fish.
As of 1999, only five drugs have been approved by the FDA for use in the aquaculture industry. Use of these drugs for particular disease conditions is highly regulated particularly regarding the use of these drugs with food fish. Formalin, oxytetracycline, and sulfadimethoxine have been approved to treat catfish diseases. Although not approved for food fish, potassium permanganate and copper sulfate are also used to treat Ick infections of ornamental fish. Other non-approved drugs, including malachite green and quinine, have been demonstrated effective against Ick. Toxicity and safety concerns have hindered FDA approval of many of these treatments. Malachite green, for example, is both mutagenic and teratogenic, and its use is restricted in many countries. Formalin, although approved, has also been demonstrated as a potential carcinogen to fish. Other drugs, such as potassium permanganate, have been demonstrated to cause gill injury at effective concentrations.
There are arguably no effective long-term treatments for fish infected with the protozoan parasite, Ichthyophthirus multifiliis. Prior treatments are ineffective in controlling the complete life cycle of Ick and have the potential to be harmful to fish or other animals, including humans. Current chemical treatments are costly and may also be labor intensive due to clean-up procedures required both before and after treatment. Last, available treatments are not practical for treating large numbers of infected fish such as those cultured in indoor hatcheries or in ponds. Phloxine B, by contrast, is non-toxic to most animal species, does not accumulate or pose an environmental threat, and is relatively inexpensive compared to other drug treatments.
The limited number of approved treatments for protozoan infections results in large production losses each year. Ick alone is responsible for nearly 50% all catfish losses reported during the Spring and Summer months of pond farm production and as mush as 80% of ornamental industry losses annually. The use of Phloxine B for treating Ick infections in fish may dramatically reduce these annual losses. Furthermore, the innovative use of a chemical currently approved by the FDA may reduce the need for extensive environmental and human safety testing before approval.
The limited number of approved and/or effective treatments for Ick infections presents a problem for the aquaculture industry. Costs, however, for approving a single therapeutic by the FDA can typically exceed $50 million. As such, pharmacological suppliers are reluctant to sponsor expensive research and testing campaigns for new drug therapy. Currently, researchers are examining alternative uses for chemicals that have already been FDA approved. For example, oxytetracycline was marketed as a human medicinal and then later approved for use in the aquaculture industry to control certain bacterial infections. Alternative uses for FDA approved chemicals can provide not only an alternative treatment for fish diseases but also reduce the costs associated with extensive FDA research and testing.
Research examining the effectiveness of photodynamic (or photoactive) dyes as pesticides has been conducted since the early seventies. The USDA""s Agricultural Research Service (USDA-ARS) have identified at least twenty photodynamic dyes that are toxic to insects, many of which are used in the human cosmetic industry. The pesticide SureDye(trademark) was discovered through a joint effort conducted by the USDA""s Animal and Plant Health Inspection Service (USDA-APHIS) and Photodye International, Inc. Specifically, studies demonstrated that Phloxine B, as the active ingredient in SureDye(trademark), was effective in the control of various Diptera species. Other photoactivated dyes such as rose Bengal and acridine orange have been shown to be toxic to fire ants and E. coli, respectively. It has been suggested that dye-light therapy may also be effective against the herpes simplex virus.
Phloxine B, is FDA approved and has been used in human cosmetics for nearly 30 years. Phloxine B exists as a powder at room temperature and melts or decomposes at higher temperatures. The compound is a halogenated xanthene dye (see FIG. 1) with a molecular weight of 691.91 g and a water solubility of greater than 120 mg/l.
Due to its hydrophilic nature, bioaccumulation of Phloxine B residues is hypothesized as unlikely. Exposure to sunlight results almost immediate degradation and detoxification ( less than 1 hr), hence the dye is not considered a potential long-term environmental hazard. Metabolites and other degradation products also appear non-toxic. According to the FDA (1982), Phloxine B is relatively non-toxic to humans when ingested (xe2x89xa61.25 mg/kg body weight). Although potentially a mild skin and eye irritant, the risk of lethal exposure to humans is unlikely. Studies performed on rats, dogs, and mice have also demonstrated the limited adverse effects of Phloxine B (LD50 (mg/kg) rat 8400, dog  greater than 4600, mouse 310).
The mechanism of action of Phloxine B is not well understood. It has been proposed that the dye collects visible light energy, converting ground state oxygen to a reactive, toxic, single oxygen molecule. Transformation of energy to the short-lived radical results in a series of reactions that eventually result in the formation of a longer-lived, metastable, triplet oxygen molecule. The excess energy of the triplet radical contributes to the oxidation of other chemicals, eventually returning the oxygen to the stable ground state. Photodynamic reactivity of Phloxine B is mediated by several physical and chemical factors including light wavelength and intensity, temperature, pH, and concentration of the dye itself. Because the transfer of light energy to oxygen is facilitated by halogens, the toxicity of the dye directly increases with increasing halogenation.
Studies have demonstrated that photoactive dyes may damage DNA, nucleotides, prokaryotic and eukaryotic cell membranes, viral membranes, and cytosolic proteins. The FDA approved photoactive compound, SureDye(trademark), is administered to insects via food baits. Although the target of toxicity is unknown, researchers suggest that external tissues, not internal organs, are the regions of immediate toxicity. It has been suggested that humans and other animals are protected from the effects of ingesting phototoxic dyes since the internal organs function in relative darkness and, if absorbed, would be rapidly eliminated by the intestines and liver.
The present Invention comprises the use of photoactive dyes such as Phloxine B, which is chemically known as 2xe2x80x2, 4xe2x80x2, 5xe2x80x2 7xe2x80x2-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein, disodium salt and which is registered as DandC (Drug and Cosmetic) Red Dye #28, as a treatment for fish infected with external protozoans such as Ichthyophthirius multifiliis. It has been discovered that photoactive dyes such as Phloxine B are readily absorbed by external protozoans such as Ichthyophthirius multifiliis. It has also been discovered that, after absorption by translucent protozoa such as Ichthyophthirius multifiliis, photoactive dyes may be photoactivated by exposing the translucent external protozoa to light. Moreover, it has been discovered that exposure of translucent protozoans to light after absorption of photoactive dyes such as Phloxine B is toxic to the protozoan, presumably due to photoactivation of the photoactive dye.
There are many advantages over the prior art of using photoactive dyes such as Phloxine B as a treatment for fish infected with external protozoans. For example, photactive dyes such as Phloxine B are non-toxic to most animal species, do not accumulate or pose an environmental threat, and are relatively inexpensive. Moreover, because exposure to sunlight results in almost immediate degradation and detoxification, any long-term environmental hazards are unlikely.
According to one embodiment of the invention, there is disclosed a method of treating protozoan infections in fish including introducing a quantity of photoactive dye, such as Phloxine B, to an aqueous environment containing one or more fish infected with protozoa. According to one aspect of the invention, the method includes introducing under low-light conditions a quantity of photoactive dye to an aqueous environment containing one or more fish infected with protozoa.
According to yet another embodiment of the invention, there is disclosed a method of treating protozoan infections in fish including introducing a quantity of photoactive dye to an aqueous environment containing one or more fish infected with protozoan such that the resulting concentration of the photoactive dye in the aqueous environment is toxic to the protozoan. According to one aspect of the invention, the method includes introducing under low-light conditions a quantity of photoactive dye to an aqueous environment containing one or more fish infected with protozoan such that the resulting concentration of the photoactive dye in the aqueous environment is toxic to the protozoan.
According to yet another embodiment of the invention, there is disclosed a method of treating protozoan infections in fish including the steps of: (a) introducing a quantity of photoactive dye to an aqueous environment containing one or more fish infected with protozoa; and (b) repeating step xe2x80x9c(a)xe2x80x9d one or more times. According to one aspect of the invention, the method includes the steps of: (a) introducing a quantity of photoactive dye under low-light conditions to an aqueous environment containing one or more fish infected with protozoa; and (b) repeating step xe2x80x9c(a)xe2x80x9d one or more times.
According to yet another embodiment of the invention, there is disclosed a method of treating protozoan infections in fish including the steps of: (a) introducing a quantity of photoactive dye to an aqueous environment containing one or more fish infected with protozoan such that the resulting concentration of the photoactive dye in the aqueous environment is toxic to the protozoan; and (b) repeating step (a) one or more times. According to one aspect of the invention, the method includes the steps of: (a) introducing a quantity of photoactive dye under low-light conditions to an aqueous environment containing one or more fish infected with protozoan such that the resulting concentration of the photoactive dye in the aqueous environment is toxic to the protozoan; and (b) repeating step (a) one or more times.
According to yet another embodiment of the invention, there is disclosed a method of treating protozoan infections in fish including the steps of: (a) introducing a quantity of photoactive dye under low-light conditions to an aqueous environment containing one or more fish infected with protozoan; (b) allowing sufficient time for protozoan absorption of the photoactive dye under low-light conditions; and (c) photoactivating the absorbed photoactive dye. According to one aspect of the invention, the method includes the steps of: (a) introducing a quantity of photoactive dye under low-light conditions to an aqueous environment containing one or more fish infected with protozoan; (b) allowing sufficient time for protozoan absorption of the photoactive dye under low-light conditions; (c) photoactivating the absorbed photoactive dye; and (d) repeating steps (a)-(c) one or more times.
In accordance with these discoveries, it is an object of the present invention to provide a method for the control of external protozoan in fish populations. Other objects of the present invention will become readily apparent from the following description.